lncTIMP3 promotes osteogenic differentiation of bone marrow mesenchymal stem cells via miR-214/Smad4 axis to relieve postmenopausal osteoporosis.

BACKGROUND: Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation plays an important regulatory role in this process. Recently, several long non-coding RNAs (lncRNAs) have been reported to play an important regulatory role in the occurrence and development of OP and participates in a variety of physiological and pathological processes. However, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIMP3) remains to be investigated. METHODS: The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIMP3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay. RESULTS: The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and Ocn were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIMP3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIMP3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIMP3 exhibited the reverse results. Subsequently, lncTIMP3 was confirmed to be located in the cytoplasm of BMSCs, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIMP3 and Smad4 were elucidated in vitro. CONCLUSION: lncTIMP3 may delay the progress of PMOP by promoting the activity of BMSC, the level of osteogenic differentiation marker gene and the formation of calcium nodules by acting on the miR-214/Smad4 axis. This finding may offer valuable insights into the possible management of PMOP.

MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

SMAD4 promotes somatic-germline contact during murine oocyte growth.

Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFß) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFß mediator, SMAD4, in mouse granulosa cells. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.

Gamma radiation assisted green synthesis of hesperidin-reduced graphene oxide nanocomposite targeted JNK/SMAD4/MMP2 signaling pathway.

In this study, a novel method for the fabrication of hesperidin/reduced graphene oxide nanocomposite (RGOH) with the assistance of gamma rays is reported. The different RGOHs were obtained by varying hesperidin concentrations (25, 50, 100, and 200 wt.%) in graphene oxide (GO) solution. Hesperidin concentrations (25, 50, 100, and 200 wt.%) in graphene oxide (GO) were varied to produce the various RGOHs. Upon irradiation with 80 kGy from γ-Ray, the successful reduction of GO occurred in the presence of hesperidin. The reduction process was confirmed by different characterization techniques such as FTIR, XRD, HRTEM, and Raman Spectroscopy. A cytotoxicity study using the MTT method was performed to evaluate the cytotoxic-anticancer effects of arbitrary RGOH on Wi38, CaCo2, and HepG2 cell lines. The assessment of RGOH's anti-inflammatory activity, including the monitoring of IL-1B and IL-6 activities as well as NF-kB gene expression was done. In addition, the anti-invasive and antimetastatic properties of RGOH, ICAM, and VCAM were assessed. Additionally, the expression of the MMP2-9 gene was quantified. The assessment of apoptotic activity was conducted by the detection of gene expressions related to BCl2 and P53. The documentation of the JNK/SMAD4/MMP2 signaling pathway was ultimately accomplished. The findings of our study indicate that RGOH therapy has significant inhibitory effects on the JNK/SMAD4/MMP2 pathway. This suggests that it could be a potential therapeutic option for cancer.

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of ß-Catenin in Breast Cancer Cell Lines.

Increased expression and nuclear translocation of ß-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting ß-CATENIN are not as efficient as desired. Therefore, detailed understanding of ß-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing ß-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of ß-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce ß-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces ß-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on ß-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced ß-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding ß-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.

Smad4 deficiency inhibits lung metastases through enhancing phagocytosis of lung interstitial macrophages.

Smad4, a critical mediator of TGF-ß signaling, plays a pivotal role in regulating various cellular functions, including immune responses. In this study, we investigated the impact of Smad4 knockout specifically in macrophages on anti-tumor immunity, focusing on lung metastasis of B16 melanoma cells. Using a mouse model with Smad4 knockout in macrophages established via Lyz2-cre mice and Smad4 flox/flox mice, we demonstrated a significant inhibition of B16 metastasis in the lungs. Interestingly, the inhibition of tumor growth was found to be independent of adaptive immunity, as no significant changes were observed in the numbers or activities of T cells, B cells, or NK cells. Instead, Smad4 knockout led to the emergence of an MCHIIlow CD206high subset of lung interstitial macrophages, characterized by enhanced phagocytosis function. Our findings highlight the crucial role of Smad4 in modulating the innate immune response against tumors and provide insights into potential therapeutic strategies targeting lung interstitial macrophages to enhance anti-tumor immunity.

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

The Epstein-Barr virus-miRNA-BART6-5p regulates TGF-ß/SMAD4 pathway to induce glycolysis and enhance proliferation and metastasis of gastric cancer cells.

Background: EBV-miR-BARTs exhibit significant relevance in epithelial tumors, particularly in EBV-associated gastric and nasopharyngeal cancers. However, their specific mechanisms in the initiation and progression of gastric cancer remain insufficiently explored. Material and Methods: Initially, EBV-miRNA-BART6-5p and its target gene SMAD4 expression were assessed in EBV-associated gastric cancer tissues and cell lines. Subsequent transfection induced overexpression of EBV-miRNA-BART6-5p in AGS and MKN-45, and downregulation in EBV-positive cells (SUN-719). The subsequent evaluation aimed to observe their impact on gastric cancer cell proliferation, migration, and glycolytic processes, with the TGF-ß/SMAD4 signaling pathway value clarified using a TGF-ß inhibitor. Results: EBV-miRNA-BART6-5p exhibits pronounced upregulation in EBV-associated gastric cancer tissues and EBV-positive cells, while its target gene SMAD4 demonstrates downregulated expression. Upregulation of it can promote the proliferation and migration of gastric cancer cells. Additionally, We found EBV-miRNA-BART6-5p promotes glycolysis of gastric cancer cells. Inhibition of the TGF-ß/SMAD4 signaling pathway resulted in suppressed proliferation and migration of gastric cancer cells, concomitant with a diminished glycolytic capacity. Conclusion: In this study, we found that EBV-miRNA-BART6-5p can target SMAD4, effectively increasing glycolysis in gastric cancer cells by regulating the TGF-ß/SMAD4 signaling pathway, thereby enhancing the proliferation and metastasis of gastric cancer cells. Our findings may offer new insights into the metabolic aspects of gastric cancer.

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4.

BACKGROUND: Pancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis. METHODS: The expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People's Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied. RESULTS: LINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models. CONCLUSION: We demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

FLRT3 and TGF-ß/SMAD4 signalling: Impacts on apoptosis, autophagy and ion channels in supraventricular tachycardia.

To explore the underlying molecular mechanisms of supraventricular tachycardia (SVT), this study aimed to analyse the complex relationship between FLRT3 and TGF-ß/SMAD4 signalling pathway, which affects Na+ and K+ channels in cardiomyocytes. Bioinformatics analysis was performed on 85 SVT samples and 15 healthy controls to screen overlapping genes from the key module and differentially expressed genes (DEGs). Expression profiling of overlapping genes, coupled with Receiver Operating Characteristic (ROC) curve analyses, identified FLRT3 as a hub gene. In vitro studies utilizing Ang II-stimulated H9C2 cardiomyocytes were undertaken to elucidate the consequences of FLRT3 silencing on cardiomyocyte apoptosis and autophagic processes. Utilizing a combination of techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting (WB), flow cytometry, dual-luciferase reporter assays and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were conducted to decipher the intricate interactions between FLRT3, the TGF-ß/SMAD4 signalling cascade and ion channel gene expression. Six genes (AADAC, DSC3, FLRT3, SYT4, PRR9 and SERTM1) demonstrated reduced expression in SVT samples, each possessing significant clinical diagnostic potential. In H9C2 cardiomyocytes, FLRT3 silencing mitigated Ang II-induced apoptosis and modulated autophagy. With increasing TGF-ß concentration, there was a dose-responsive decline in FLRT3 and SCN5A expression, while both KCNIP2 and KCND2 expressions were augmented. Moreover, a direct interaction between FLRT3 and SMAD4 was observed, and inhibition of SMAD4 expression resulted in increased FLRT3 expression. Our results demonstrated that the TGF-ß/SMAD4 signalling pathway plays a critical role by regulating FLRT3 expression, with potential implications for ion channel function in SVT.

The immune regulation and therapeutic potential of the SMAD gene family in breast cancer.

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

Mutational profile evaluates metastatic capacity of Chinese colorectal cancer patients, revealed by whole-exome sequencing.

Colorectal cancer (CRC) is the third most common cancer and the prevalence rate of CRC is increasing in the China. In this study, whole-exome sequencing (WES) was performed on primary tissues of 47 CRC Chinese patients including 22 metastatic and 25 non-metastatic patients. By comparison with data from western colorectal cancer patients in the Cancer Genome Atlas (TCGA), we identified a number of genes that are more likely to be mutated in Chinese colorectal cancer patients, such as MUC12, MUC12, MUC2, MUC4, HYDIN and KMT2C. Interestingly, MUC family genes including MUC12, MUC2 and MUC4, have mutation rates of >20%, while the mutation frequency was extremely low in western colorectal cancer patients, which were <3% in TCGA and 0% in Memorial Sloan Kettering Cancer Center (MSKCC). We detected metastasis-specific mutated genes including TCF7L2, MST1L, HRNR and SMAD4, while MUC4, NEB, FLG and RFPL4A alteration is more prevalent in the non-metastasis group. Further analysis reveals mutation genes in metastasis group are more focus in the Wnt and Hippo signaling pathway. APC, SMAD4 and TCF7L2 accounted for the major genetic abnormalities in this pathway. In conclusion, this study identified the unique characteristics of gene mutations in Chinese patients with colorectal cancer, and is a valuable reference for personalized treatment in Chinese CRC patients.

Dantrolene and coenzyme Q10 as a suggested combination therapy to statin-induced myopathy in high fat diet rats: A possible interference with ROS/ TGF-ß / Smad4 signaling pathway.

One of the major hitches for statins' utilization is the development of myotoxicity. Versatile studies reported that the underlining molecular mechanisms including coenzyme Q10 (CoQ10)/ubiquinone depletion, as well as the disturbance in the cytoplasmic Ca2+ homeostasis. Therefore, we investigated the consequences of supplementing CoQ10 and dantrolene, a cytoplasmic Ca2+ reducing agent, in combination with simvastatin. This adjuvant therapy normalized the simvastatin-mediated elevation in serum ALT, AST, CK-MM, as well as tissue Ca2+ content, in addition to suppressing the simvastatin-mediated oxidative stress in simvastatin-treated rats, while having no effect upon statin-induced antihyperlipidemic effect. Additionally, the combination inhibited the simvastatin-induced TGF-ß/ Smad4 pathway activation. Collectively, the current study emphasizes on the potential utilization of dantrolene and CoQ10 as an adjuvant therapy to statins treatment for improving their side effect profile.

HBO1 determines SMAD action in pluripotency and mesendoderm specification.

TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.

Reducing SULT2B1 promotes the interaction of LncRNAgga3-204 with SMAD4 to inhibit the macrophage inflammatory response and delay atherosclerosis progression.

Inflammation is a crucial pathophysiological mechanism in atherosclerosis (AS). This study aims to investigate the impact of sulfotransferase family 2b member 1 (SULT2B1) on the inflammatory response of macrophages and the progression of AS. Here, we reported that SULT2B1 expression increased with the progression of AS. In AS model mice, knockdown of Sult2b1 led to remission of AS and reduced inflammation levels. Further exploration of the downstream molecular mechanisms of SULT2B1 revealed that suppressing Sult2b1 in macrophages resulted in decreased levels of 25HC3S in the nucleus, elevated expression of Lxr, and increased the transcription of Lncgga3-204. In vivo, knockdown of Lncgga3-204 aggravated the inflammatory response and AS progression, while the simultaneous knockdown of both Sult2b1 and Lncgga3-204 exacerbated AS and the inflammatory response compared with knockdown of Sult2b1 alone. Increased binding of Lncgga3-204 to SMAD4 in response to oxidized-low density lipoprotein (ox-LDL) stimulation facilitated SMAD4 entry into the nucleus and regulated Smad7 transcription, which elevated SMAD7 expression, suppressed NF-κB entry into the nucleus, and ultimately attenuated the macrophage inflammatory response. Finally, we identified the presence of a single nucleotide polymorphism (SNP), rs2665580, in the SULT2B1 promoter region in monocytes from coronary artery disease (CAD) patients. The predominant GG/AG/AA genotypes were observed in the Asian population. Elevated SULT2B1 expression in monocytes with GG corresponded to elevated inflammatory factor levels and more unstable coronary plaques. To summarize, our study demonstrated that the critical role of SULT2B1/Lncgga3-204/SMAD4/NF-κB in AS progression. SULT2B1 serves as a novel biomarker indicating inflammatory status, thereby offering insights into potential therapeutic strategies for AS.

miR-6881-3p contributes to diminished ovarian reserve by regulating granulosa cell apoptosis by targeting SMAD4.

BACKGROUND: In our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood. OBJECTIVE: This study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR. MATERIALS AND METHODS: Initially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes. RESULTS: Our findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3'-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased. CONCLUSION: This study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.

SMAD4 endows TGF-ß1-induced highly invasive tumor cells with ferroptosis vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy prone to recurrence and metastasis. Studies show that tumor cells with increased invasive and metastatic potential are more likely to undergo ferroptosis. SMAD4 is a critical molecule in the transforming growth factor ß (TGF-ß) pathway, which affects the TGF-ß-induced epithelial-mesenchymal transition (EMT) status. SMAD4 loss is observed in more than half of patients with PDAC. In this study, we investigated whether SMAD4-positive PDAC cells were prone to ferroptosis because of their high invasiveness. We showed that SMAD4 status almost determined the orientation of transforming growth factor ß1 (TGF-ß1)-induced EMT via the SMAD4-dependent canonical pathway in PDAC, which altered ferroptosis vulnerability. We identified glutathione peroxidase 4 (GPX4), which inhibited ferroptosis, as a SMAD4 down-regulated gene by RNA sequencing. We found that SMAD4 bound to the promoter of GPX4 and decreased GPX4 transcription in PDAC. Furthermore, TGF-ß1-induced high invasiveness enhanced sensitivity of SMAD4-positive organoids and pancreas xenograft models to the ferroptosis inducer RAS-selective lethal 3 (RSL3). Moreover, SMAD4 enhanced the cytotoxic effect of gemcitabine combined with RSL3 in highly invasive PDAC cells. This study provides new ideas for the treatment of PDAC, especially SMAD4-positive PDAC.

Sporadic gastric juvenile polyposis with a novel SMAD4 nonsense mutation in a mosaic pattern.

A 50-year-old female was diagnosed with gastric hyperplastic polyps 7 years before and was followed up at another hospital. She was referred to our hospital because of the growth of gastric polyps and progression of anemia. She had no family history of polyposis. The polyps were observed only in the stomach, increased in size and number, and the erythematous edema got worse. Endoscopic mucosal resection (EMR) of the gastric polyp was performed. Pathologically, the gastric polyp was hamartomatous polyp, and the intervening mucosa between polyps showed no atypical structure without inflammation. Given that gastric juvenile polyposis (GJP) was clinically suspected, a genetic test using peripheral blood was performed. Target resequencing and Sanger sequencing analysis revealed a nonsense mutation in the SMAD4 gene at codon 169. The mutation was detected at a low frequency of 11%, and considered a mosaic mutation. Therefore, she was diagnosed with a sporadic GJP, and total gastrectomy was performed. Immunostaining of SMAD4 for the resected specimen showed a mixture of stained and unstained area in the epithelium of the polyp, indicating partial loss of SMAD4 expression. To our knowledge, this is the first reported case of GJP with a nonsense SMAD4 mutation at codon 169 in a mosaic pattern.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies.

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.